The importance of Cre recombinase to minimize helper vector (HV) contamination during helper-depe... more The importance of Cre recombinase to minimize helper vector (HV) contamination during helper-dependent adenovirus vectors (HDVs) production is well documented. However, Cre recombinase, by inducing DNA double-strand breaks (DSBs), can cause a reduced proliferation and genotoxic effects in cultured cells. In this work, Cre-expressing cell stability, co-infection and their relation to adenovirus amplification/HV contamination were evaluated to develop a production protocol for HD canine adenovirus type 2 (CAV-2) vectors. Long-term Cre expression reduced the capacity of MDCK-E1-Cre cells to produce CAV-2 by 7-fold, although cell growth was maintained. High HDV/HV MOI ratio (550.1) led to low HV contamination without compromising HDV yields. Indeed, such MOI ratio was sufficient to reduce HV levels, as these were similar either in MDCK-E1 or MDCK-E1-Cre cells. This raises the possibility of producing HDVs without Cre-expressing cells, which would circumvent the negative effects that this recombinase holds to the production system. Here, we show how Cre and MOI ratio impact adenovirus vectors yields and infectivity, providing key-information to design an improved manufacturing of HDV. Potential mechanisms to explain how Cre is specifically impacting cell productivity without critically compromising its growth are presented. OPEN SUBJECT AREAS: GENE THERAPY CELL BIOLOGY ADENOVIRUS MOLECULAR BIOLOGY
The productivity of retroviral vector producer cell lines is currently low; this can be attribute... more The productivity of retroviral vector producer cell lines is currently low; this can be attributed partly to disruption of the viral components stoichiometry originated by the dissociation of gag-pol, env, and viral genome. This paper addresses the impact of viral components stoichiometry on cell productivity and vector quality (vector stability and transduction efficiency). A strategy is proposed for the development of packaging cell lines allowing for stoichiometric optimization of viral components. This strategy is based on the introduction of a transgene in the first step of cell line development, thus making it possible to eliminate transgene limitations and therefore exploring maximal infectious productivity based on the levels of expression of Gag-Pol and envelope. The transgene can subsequently be exchanged for a gene of interest, using site-specific flipase/flipase recombination target (Flp/FRT) cassette replacement. It was observed that Gag-Pol controlled the order of magnitude of infectious titer: a small, 2-fold variation in its expression could result in a 10-to 100-fold improvement in infectious titer; the RNA transgene and envelope expression limitations had a lower impact. The ratios of retroviral components strongly affected cell productivity but did not affect vector stability; however, transduction efficiencies were affected by an imbalanced ratio of the components, resulting in the production of higher amounts of defective virus. Thus, stoichiometric optimization of viral components not only improves cell productivity but also increases vector quality.
Retroviral vectors are presently amongst the most widely used vectors in gene therapy clinical tr... more Retroviral vectors are presently amongst the most widely used vectors in gene therapy clinical trials to target pathologies of different origins, such as cancers, genetic diseases or neurological disorders. This review provides an overview on the evolution of retroviral vector design and production for gene therapy applications, including state of the art developments in flexible producer cells and safe vectors. In addition, production and purification processes will be addressed, with a particular focus on the improvements undertaken to increase vector productivity and to reduce the rapid loss of infectivity, which presently represent the main challenges in retroviral vectors production for gene therapy.
The past decade witnessed the entry into the market of new virus-based biopharmaceuticals produce... more The past decade witnessed the entry into the market of new virus-based biopharmaceuticals produced in animal cells such as oncolytic vectors, virus-like particle vaccines, and gene transfer vectors. Therefore, increased attention and investment to optimize cell culture processes towards enhanced manufacturing of these bioproducts is anticipated. Herein, we review key findings on virus-host interactions that have been explored in cell culture optimization. Approaches supporting improved productivity or quality of vector preparations are discussed, mainly focusing on medium design and genetic manipulation. This review provides an integrated outline for current and future efforts in exploring cellular targets for the optimization of cell culture manufacturing of virus-based biopharmaceuticals.
Adenovirus vectors have been extensively studied through the manipulation of viral genome. Howeve... more Adenovirus vectors have been extensively studied through the manipulation of viral genome. However, little attention is being paid to their producer cell-lines; cells are selected according to virus yields, neglecting the expression profile of transcomplementing gene products underlying cell performance. This work evaluates the impact of E1 (E1A and E1B) and Cre recombinase levels in the production of E1-deleted and helper-dependent canine adenovirus type 2 (CAV-2) vectors using MDCK cells. E1A and E1B gene expression and Cre activity were evaluated in different cell clones and compared with the corresponding cell productivity and susceptibility to oxidative stress injury. CAV-2 production was proportional to E1A expression (the highest levels of E1A corresponding to productivities of 3000-5000 I.P./cell), while E1B prolonged host cell viability after infection, conferring protection against apoptosis. Cre recombinase counteracted E1B anti-apoptotic properties, however viral production was maintained under high levels of Cre. Yet, Cre recombinase side effects can be reduced using cell lines with lower Cre-activities, without compromising the excision efficiency of helper vector packaging signal. These results highlight the influence of transcomplementing gene products on CAV-2 producer cell line performance, and the ability to express high levels of E1A and E1B as an important feature for cell line establishment and high adenovirus titers. Citation: Fernandes P, Santiago VM, Rodrigues AF, Tomás H, Kremer EJ, et al. (2013) Impact of E1 and Cre on Adenovirus Vector Amplification: Developing MDCK CAV-2-E1 and E1-Cre Transcomplementing Cell Lines. PLoS ONE 8(4): e60342.
Currently, retroviral vector producer cell lines must be established for the production of each g... more Currently, retroviral vector producer cell lines must be established for the production of each gene vector. This is done by transfection of a packaging cell line with the gene of interest. In order to find a high-titer retroviral vector producer clone, exhaustive clone screening is necessary, as the random integration of the transgene gives rise to different expression levels. We established a virus producing packaging cell line, the 293 FLEX, in which the viral vector is flanked by two different FRT sites and a selection trap. Using Flp recombinase mediated cassette exchange; this vector can be replaced by another compatible retroviral vector. The first step was the tagging of 293 cells with a lacZ reporter gene, which allowed screening and choosing a high expressing chromosomal locus. After checking that, a single copy of the construct was integrated, cassette exchangeability was confirmed with a reporter targeting construct. Subsequently gag-pol and GaLV envelope genes were stably transfected. The lacZ transgene was replaced by a GFP transgene and the 293 FLEX producer cell line maintained the titer, thus validating the flexibility and efficacy of this producer cell line. The tagged retroviral producer cell clone should constitute a highly advantageous cell line since it has a predictable titer and can be rapidly used for different therapeutic applications.
Previous papers concerning the inactivation of retroviral vectors have focused either the kinetic... more Previous papers concerning the inactivation of retroviral vectors have focused either the kinetic aspects of the inactivation process or the factors affecting vector stability. The present work aims at bridging this previous knowledge by simultaneously studying inactivation kinetics and structural aspects of retroviral vectors. For this purpose, two human derived cell lines (TE Fly cells) producing retroviral vectors with amphotropic
Retrovirus vectors derived from moloney murine leukemia virus (MoMLV) were the first class of vir... more Retrovirus vectors derived from moloney murine leukemia virus (MoMLV) were the first class of viral vectors developed for gene therapy. They have been extensively used in clinical trials, particularly in ex vivo transduction of hematopoietic stem cells. Although there is a vast experience acquired with retroviruses, their manufacturing is still a difficult task due to the low cell productivities and inherent instability of the infective virus. These viral vectors are most commonly produced using stable producer cell lines in adherent monolayer culture systems. In order to obtain high transduction efficiencies and low toxicity in clinical applications, the viral preparations should be purified, concentrated, and well characterized to attain stringent quality specifications. This chapter describes currently used protocols for manufacturing retroviruses.
Traditional cell line development is quite laborious and time-consuming as it is based on the ran... more Traditional cell line development is quite laborious and time-consuming as it is based on the random integration of the gene of interest which leads to unpredictable expression behavior. In opposition, recombinase-mediated cassette exchange systems represent a powerful genetic engineering approach, allowing site-specific insertion of recombinant genes into pre-tagged genomic loci with superior expression characteristics, thus bypassing the need for extensive clone screening and shortening the development timelines. Such systems have not been widely implemented in insect cell lines used for the production of recombinant proteins most commonly through the baculovirus expression vector system. Herein, it is provided the protocol for the implementation of a FLP-mediated cassette exchange system in Spodoptera frugiperda Sf 9 cells, in order to grant a flexible cell line for the stable production of recombinant proteins.
Helper-dependent adenovirus vectors (HDVs) are safe and efficient tools for gene transfer with hi... more Helper-dependent adenovirus vectors (HDVs) are safe and efficient tools for gene transfer with high cloning capacity. However, the multiple amplification steps needed to produce HDVs hamper a robust production process and in turn the availability of high-quality vectors. To understand the factors behind the low productivity, we analyzed the progression of HDV life cycle. Canine adenovirus (Ad) type 2 vectors, holding attractive features to overcome immunogenic concerns and treat neurobiological disorders, were the focus of this work. When compared with E1-deleted (ΔE1) vectors, we found a faster helper genome replication during HDV production. This was consistent with an upregulation of the Ad polymerase and pre-terminal protein and led to higher and earlier expression of structural proteins. Although genome packaging occurred similarly to ΔE1 vectors, more immature capsids were obtained during HDV production, which led to a~4-fold increase in physical-to-infectious particles ratio. The higher viral protein content in HDV-producing cells was also consistent with an increased activation of autophagy and cell death, in which earlier cell death compromised volumetric productivity. The increased empty capsids and earlier cell death found in HDV production may partially contribute to the lower vector infectivity. However, an HDV-specific factor responsible for a defective maturation process should be also involved to fully explain the low infectious titers. This study showed how a deregulated Ad cycle progression affected cell line homeostasis and HDV propagation, highlighting the impact of vector genome design on virus-cell interaction. Impact of adenovirus life cycle on HDV generation P Fernandes et al Impact of adenovirus life cycle on HDV generation P Fernandes et al Impact of adenovirus life cycle on HDV generation P Fernandes et al Impact of adenovirus life cycle on HDV generation P Fernandes et al
Carboxylesterases are important enzymes for xenobiotic metabolism and are receiving increasing at... more Carboxylesterases are important enzymes for xenobiotic metabolism and are receiving increasing attention in the context of cancer therapies. Quantification of individual carboxylesterase activity is important since protein levels do not always correlate to activity and significant interorgan, interindividual, and interspecies variations exist. Here we present a methodology enabling the specific quantification of carboxylesterase 2 activity in a pool of other esterases. Method applicability is illustrated for the evaluation of interspecies variation and for activity assessment of transfected cell extracts. The methodology can easily be adapted to the evaluation of other esterases upon careful selection of adequate substrates and/or specific inhibitors.
A new strategy was developed that provides well-defined high-titer producer cells for recombinant... more A new strategy was developed that provides well-defined high-titer producer cells for recombinant retroviruses in a minimum of time. The strategy involves the targeted integration of the retroviral vector into a chromosomal locus with favourable properties. For proof of concept we established a novel HEK293 based retroviral producer cell line, called Flp293A, with a single copy retroviral vector integrated in a selected chromosomal locus. The vector was flanked by non-interacting Flp-recombinase recognition sites (FRT) and was exchanged for different retroviral vectors via Flp mediated cassette exchange. All analyzed cell clones showed correct integration and identical titers for each of the vectors, confirming that the expression characteristics form the parental cell were preserved. Titers up to 2.5 x10 7 ip/10 6 cells were obtained. Also, high titer producer cells for a therapeutic vector that encodes the 8.9 kb collagen VII cDNA in a marker free cassette was received within three weeks without screening.
Lentiviral vectors (LVs) hold great potential as gene delivery vehicles. However, the manufacturi... more Lentiviral vectors (LVs) hold great potential as gene delivery vehicles. However, the manufacturing and purification of these vectors still present major challenges, mainly because of the low stability of the virus, essentially due to the fragility of the membrane envelope. The main goal of this work was the establishment of a fast, scalable, and robust downstream protocol for LVs, combining microfiltration, anion-exchange, and ultrafiltration membrane technologies toward maximization of infectious LVs recovery. CIM Ò (Convective Interaction Media) monolithic columns with diethylaminoethanol (DEAE) anion exchangers were used for the purification of clarified LV supernatants, allowing infectious vector recoveries of 80%, which is 10% higher than the values currently reported in the literature. These recoveries, combined with the results obtained after optimization of the remaining downstream purification steps, resulted in overall infectious LV yields of 36%. Moreover, the inclusion of a Benzonase step allowed a removal of approximately 99% of DNA impurities. The entire downstream processing strategy herein described was conceived based on disposable and easily scalable technologies. Overall, CIM DEAE columns have shown to be a good alternative for the purification of LVs, since they allow faster processing of the viral bulks and enhanced preservation of virus biological activity, consequently, increasing infectious vector recoveries.
Proceedings of the 21st Annual Meeting of the European Society for Animal Cell Technology (ESACT), Dublin, Ireland, June 7-10, 2009, 2011
Adenovirus vectors are one of the most efficient vehicles for delivering nucleic acids into mamma... more Adenovirus vectors are one of the most efficient vehicles for delivering nucleic acids into mammalian cells. However, human adenoviruses are ubiquitous in all population, posing memory immunity responses obstacles for their use during clinical gene transfer. To ...
The importance of Cre recombinase to minimize helper vector (HV) contamination during helper-depe... more The importance of Cre recombinase to minimize helper vector (HV) contamination during helper-dependent adenovirus vectors (HDVs) production is well documented. However, Cre recombinase, by inducing DNA double-strand breaks (DSBs), can cause a reduced proliferation and genotoxic effects in cultured cells. In this work, Cre-expressing cell stability, co-infection and their relation to adenovirus amplification/HV contamination were evaluated to develop a production protocol for HD canine adenovirus type 2 (CAV-2) vectors. Long-term Cre expression reduced the capacity of MDCK-E1-Cre cells to produce CAV-2 by 7-fold, although cell growth was maintained. High HDV/HV MOI ratio (550.1) led to low HV contamination without compromising HDV yields. Indeed, such MOI ratio was sufficient to reduce HV levels, as these were similar either in MDCK-E1 or MDCK-E1-Cre cells. This raises the possibility of producing HDVs without Cre-expressing cells, which would circumvent the negative effects that this recombinase holds to the production system. Here, we show how Cre and MOI ratio impact adenovirus vectors yields and infectivity, providing key-information to design an improved manufacturing of HDV. Potential mechanisms to explain how Cre is specifically impacting cell productivity without critically compromising its growth are presented. OPEN SUBJECT AREAS: GENE THERAPY CELL BIOLOGY ADENOVIRUS MOLECULAR BIOLOGY
The productivity of retroviral vector producer cell lines is currently low; this can be attribute... more The productivity of retroviral vector producer cell lines is currently low; this can be attributed partly to disruption of the viral components stoichiometry originated by the dissociation of gag-pol, env, and viral genome. This paper addresses the impact of viral components stoichiometry on cell productivity and vector quality (vector stability and transduction efficiency). A strategy is proposed for the development of packaging cell lines allowing for stoichiometric optimization of viral components. This strategy is based on the introduction of a transgene in the first step of cell line development, thus making it possible to eliminate transgene limitations and therefore exploring maximal infectious productivity based on the levels of expression of Gag-Pol and envelope. The transgene can subsequently be exchanged for a gene of interest, using site-specific flipase/flipase recombination target (Flp/FRT) cassette replacement. It was observed that Gag-Pol controlled the order of magnitude of infectious titer: a small, 2-fold variation in its expression could result in a 10-to 100-fold improvement in infectious titer; the RNA transgene and envelope expression limitations had a lower impact. The ratios of retroviral components strongly affected cell productivity but did not affect vector stability; however, transduction efficiencies were affected by an imbalanced ratio of the components, resulting in the production of higher amounts of defective virus. Thus, stoichiometric optimization of viral components not only improves cell productivity but also increases vector quality.
Retroviral vectors are presently amongst the most widely used vectors in gene therapy clinical tr... more Retroviral vectors are presently amongst the most widely used vectors in gene therapy clinical trials to target pathologies of different origins, such as cancers, genetic diseases or neurological disorders. This review provides an overview on the evolution of retroviral vector design and production for gene therapy applications, including state of the art developments in flexible producer cells and safe vectors. In addition, production and purification processes will be addressed, with a particular focus on the improvements undertaken to increase vector productivity and to reduce the rapid loss of infectivity, which presently represent the main challenges in retroviral vectors production for gene therapy.
The past decade witnessed the entry into the market of new virus-based biopharmaceuticals produce... more The past decade witnessed the entry into the market of new virus-based biopharmaceuticals produced in animal cells such as oncolytic vectors, virus-like particle vaccines, and gene transfer vectors. Therefore, increased attention and investment to optimize cell culture processes towards enhanced manufacturing of these bioproducts is anticipated. Herein, we review key findings on virus-host interactions that have been explored in cell culture optimization. Approaches supporting improved productivity or quality of vector preparations are discussed, mainly focusing on medium design and genetic manipulation. This review provides an integrated outline for current and future efforts in exploring cellular targets for the optimization of cell culture manufacturing of virus-based biopharmaceuticals.
Adenovirus vectors have been extensively studied through the manipulation of viral genome. Howeve... more Adenovirus vectors have been extensively studied through the manipulation of viral genome. However, little attention is being paid to their producer cell-lines; cells are selected according to virus yields, neglecting the expression profile of transcomplementing gene products underlying cell performance. This work evaluates the impact of E1 (E1A and E1B) and Cre recombinase levels in the production of E1-deleted and helper-dependent canine adenovirus type 2 (CAV-2) vectors using MDCK cells. E1A and E1B gene expression and Cre activity were evaluated in different cell clones and compared with the corresponding cell productivity and susceptibility to oxidative stress injury. CAV-2 production was proportional to E1A expression (the highest levels of E1A corresponding to productivities of 3000-5000 I.P./cell), while E1B prolonged host cell viability after infection, conferring protection against apoptosis. Cre recombinase counteracted E1B anti-apoptotic properties, however viral production was maintained under high levels of Cre. Yet, Cre recombinase side effects can be reduced using cell lines with lower Cre-activities, without compromising the excision efficiency of helper vector packaging signal. These results highlight the influence of transcomplementing gene products on CAV-2 producer cell line performance, and the ability to express high levels of E1A and E1B as an important feature for cell line establishment and high adenovirus titers. Citation: Fernandes P, Santiago VM, Rodrigues AF, Tomás H, Kremer EJ, et al. (2013) Impact of E1 and Cre on Adenovirus Vector Amplification: Developing MDCK CAV-2-E1 and E1-Cre Transcomplementing Cell Lines. PLoS ONE 8(4): e60342.
Currently, retroviral vector producer cell lines must be established for the production of each g... more Currently, retroviral vector producer cell lines must be established for the production of each gene vector. This is done by transfection of a packaging cell line with the gene of interest. In order to find a high-titer retroviral vector producer clone, exhaustive clone screening is necessary, as the random integration of the transgene gives rise to different expression levels. We established a virus producing packaging cell line, the 293 FLEX, in which the viral vector is flanked by two different FRT sites and a selection trap. Using Flp recombinase mediated cassette exchange; this vector can be replaced by another compatible retroviral vector. The first step was the tagging of 293 cells with a lacZ reporter gene, which allowed screening and choosing a high expressing chromosomal locus. After checking that, a single copy of the construct was integrated, cassette exchangeability was confirmed with a reporter targeting construct. Subsequently gag-pol and GaLV envelope genes were stably transfected. The lacZ transgene was replaced by a GFP transgene and the 293 FLEX producer cell line maintained the titer, thus validating the flexibility and efficacy of this producer cell line. The tagged retroviral producer cell clone should constitute a highly advantageous cell line since it has a predictable titer and can be rapidly used for different therapeutic applications.
Previous papers concerning the inactivation of retroviral vectors have focused either the kinetic... more Previous papers concerning the inactivation of retroviral vectors have focused either the kinetic aspects of the inactivation process or the factors affecting vector stability. The present work aims at bridging this previous knowledge by simultaneously studying inactivation kinetics and structural aspects of retroviral vectors. For this purpose, two human derived cell lines (TE Fly cells) producing retroviral vectors with amphotropic
Retrovirus vectors derived from moloney murine leukemia virus (MoMLV) were the first class of vir... more Retrovirus vectors derived from moloney murine leukemia virus (MoMLV) were the first class of viral vectors developed for gene therapy. They have been extensively used in clinical trials, particularly in ex vivo transduction of hematopoietic stem cells. Although there is a vast experience acquired with retroviruses, their manufacturing is still a difficult task due to the low cell productivities and inherent instability of the infective virus. These viral vectors are most commonly produced using stable producer cell lines in adherent monolayer culture systems. In order to obtain high transduction efficiencies and low toxicity in clinical applications, the viral preparations should be purified, concentrated, and well characterized to attain stringent quality specifications. This chapter describes currently used protocols for manufacturing retroviruses.
Traditional cell line development is quite laborious and time-consuming as it is based on the ran... more Traditional cell line development is quite laborious and time-consuming as it is based on the random integration of the gene of interest which leads to unpredictable expression behavior. In opposition, recombinase-mediated cassette exchange systems represent a powerful genetic engineering approach, allowing site-specific insertion of recombinant genes into pre-tagged genomic loci with superior expression characteristics, thus bypassing the need for extensive clone screening and shortening the development timelines. Such systems have not been widely implemented in insect cell lines used for the production of recombinant proteins most commonly through the baculovirus expression vector system. Herein, it is provided the protocol for the implementation of a FLP-mediated cassette exchange system in Spodoptera frugiperda Sf 9 cells, in order to grant a flexible cell line for the stable production of recombinant proteins.
Helper-dependent adenovirus vectors (HDVs) are safe and efficient tools for gene transfer with hi... more Helper-dependent adenovirus vectors (HDVs) are safe and efficient tools for gene transfer with high cloning capacity. However, the multiple amplification steps needed to produce HDVs hamper a robust production process and in turn the availability of high-quality vectors. To understand the factors behind the low productivity, we analyzed the progression of HDV life cycle. Canine adenovirus (Ad) type 2 vectors, holding attractive features to overcome immunogenic concerns and treat neurobiological disorders, were the focus of this work. When compared with E1-deleted (ΔE1) vectors, we found a faster helper genome replication during HDV production. This was consistent with an upregulation of the Ad polymerase and pre-terminal protein and led to higher and earlier expression of structural proteins. Although genome packaging occurred similarly to ΔE1 vectors, more immature capsids were obtained during HDV production, which led to a~4-fold increase in physical-to-infectious particles ratio. The higher viral protein content in HDV-producing cells was also consistent with an increased activation of autophagy and cell death, in which earlier cell death compromised volumetric productivity. The increased empty capsids and earlier cell death found in HDV production may partially contribute to the lower vector infectivity. However, an HDV-specific factor responsible for a defective maturation process should be also involved to fully explain the low infectious titers. This study showed how a deregulated Ad cycle progression affected cell line homeostasis and HDV propagation, highlighting the impact of vector genome design on virus-cell interaction. Impact of adenovirus life cycle on HDV generation P Fernandes et al Impact of adenovirus life cycle on HDV generation P Fernandes et al Impact of adenovirus life cycle on HDV generation P Fernandes et al Impact of adenovirus life cycle on HDV generation P Fernandes et al
Carboxylesterases are important enzymes for xenobiotic metabolism and are receiving increasing at... more Carboxylesterases are important enzymes for xenobiotic metabolism and are receiving increasing attention in the context of cancer therapies. Quantification of individual carboxylesterase activity is important since protein levels do not always correlate to activity and significant interorgan, interindividual, and interspecies variations exist. Here we present a methodology enabling the specific quantification of carboxylesterase 2 activity in a pool of other esterases. Method applicability is illustrated for the evaluation of interspecies variation and for activity assessment of transfected cell extracts. The methodology can easily be adapted to the evaluation of other esterases upon careful selection of adequate substrates and/or specific inhibitors.
A new strategy was developed that provides well-defined high-titer producer cells for recombinant... more A new strategy was developed that provides well-defined high-titer producer cells for recombinant retroviruses in a minimum of time. The strategy involves the targeted integration of the retroviral vector into a chromosomal locus with favourable properties. For proof of concept we established a novel HEK293 based retroviral producer cell line, called Flp293A, with a single copy retroviral vector integrated in a selected chromosomal locus. The vector was flanked by non-interacting Flp-recombinase recognition sites (FRT) and was exchanged for different retroviral vectors via Flp mediated cassette exchange. All analyzed cell clones showed correct integration and identical titers for each of the vectors, confirming that the expression characteristics form the parental cell were preserved. Titers up to 2.5 x10 7 ip/10 6 cells were obtained. Also, high titer producer cells for a therapeutic vector that encodes the 8.9 kb collagen VII cDNA in a marker free cassette was received within three weeks without screening.
Lentiviral vectors (LVs) hold great potential as gene delivery vehicles. However, the manufacturi... more Lentiviral vectors (LVs) hold great potential as gene delivery vehicles. However, the manufacturing and purification of these vectors still present major challenges, mainly because of the low stability of the virus, essentially due to the fragility of the membrane envelope. The main goal of this work was the establishment of a fast, scalable, and robust downstream protocol for LVs, combining microfiltration, anion-exchange, and ultrafiltration membrane technologies toward maximization of infectious LVs recovery. CIM Ò (Convective Interaction Media) monolithic columns with diethylaminoethanol (DEAE) anion exchangers were used for the purification of clarified LV supernatants, allowing infectious vector recoveries of 80%, which is 10% higher than the values currently reported in the literature. These recoveries, combined with the results obtained after optimization of the remaining downstream purification steps, resulted in overall infectious LV yields of 36%. Moreover, the inclusion of a Benzonase step allowed a removal of approximately 99% of DNA impurities. The entire downstream processing strategy herein described was conceived based on disposable and easily scalable technologies. Overall, CIM DEAE columns have shown to be a good alternative for the purification of LVs, since they allow faster processing of the viral bulks and enhanced preservation of virus biological activity, consequently, increasing infectious vector recoveries.
Proceedings of the 21st Annual Meeting of the European Society for Animal Cell Technology (ESACT), Dublin, Ireland, June 7-10, 2009, 2011
Adenovirus vectors are one of the most efficient vehicles for delivering nucleic acids into mamma... more Adenovirus vectors are one of the most efficient vehicles for delivering nucleic acids into mammalian cells. However, human adenoviruses are ubiquitous in all population, posing memory immunity responses obstacles for their use during clinical gene transfer. To ...
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